2,5-DKG permeases

ABSTRACT

The invention provides isolated nucleic acid molecules encoding polypeptides having 2,5-DKG permease activity, and oligonucleotides therefrom. The isolated nucleic acid molecules can be expressed in appropriate bacterial cells to enhance the production of 2-KLG, which can subsequently be converted to ascorbic acid. Further provided are isolated polypeptides having 2,5-DKG permease activity, immunogenic peptides therefrom, and antibodies specific therefor. The invention also provides methods of identifying novel 2,5-DKG permeases.

This application is a Divisional Application of U.S. application Ser.No. 09/922,501, which was filed Aug. 3, 2001, now U.S. Pat. No.6,720,168 and which claims the benefit of U.S. Provisional ApplicationSer. No. 60/325,774, filed Aug. 4, 2000, which was converted from U.S.Ser. No. 09/633,294 and U.S. Provisional Application Ser. No. 60/421,141, filed Sep. 29, 2000, which was converted from U.S. Ser. No.09/677,032, each of which is incorporated herein by reference in itsentirety.

This invention was made in part with U.S. Government support underCooperative Agreement 70NANB5H1138 and ATP NIST project IdentificationNumber 1995-05-0007E. The U.S. Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to microbial transporterproteins and, more specifically, to novel 2,5-diketo-D-gluconic acid(2,5-DKG) permeases.

2. Background Information

Adequate intake of ascorbic acid, or vitamin C, is recognized as animportant factor in maintaining health. To ensure adequate intake ofascorbic acid, the chemical is now added to many foods, drinks andcosmetic products, and is also sold as a direct vitamin supplement. Tomeet the commercial demand for ascorbic acid, there is a need to developmore efficient processes for its production.

Although there are a number of alternative methods of producing ascorbicacid, one of the least expensive and most ecologically sound methods isbiofermentation. Bacterial strains have now been engineered to expressall of the enzymes required for the stepwise conversion of aninexpensive sugar source, such as D-glucose, to a stable precursor ofascorbic acid, 2-keto-L-gulonic acid (2-KLG) (see U.S. Pat. No.5,032,514 and references therein). 2-KLG can be readily converted toascorbic acid by chemical or enzymatic procedures.

FIG. 2 shows schematically the enzymatic reactions that take place inthe bioconversion of D-glucose to 2-KLG. As shown in FIG. 2, theenzymatic reactions that lead from D-glucose, to D-gluconic acid, to2-keto-D-gluconic acid (2-KDG), to 2,5-diketo-D-gluconic acid (2,5-DKG),take place at the surface of the bacterial cell. 2,5-DKG must then enterthe cell in order for its enzymatic conversion to 2-KLG.

Much effort has been expended in increasing the efficiency of theenzymatic reactions involved in 2-KLG production. For example, U.S. Pat.No. 5,032,514 describes methods for increasing 2-KLG production byreducing metabolic diversion of 2,5-DKG to products other than 2-KLG.

Increasing uptake of 2,5-DKG by a bacterial strain suitable forbiofermentation could be advantageous in increasing 2-KLG production.Expressing additional copies of an endogenous 2,5-DKG permease, orexpressing an exogenous 2,5-DKG permease with superior properties, couldincrease uptake of 2,5-DKG. However, to date, no 2,5-DKG permease hasbeen identified or characterized that could be used in this manner.

Therefore, there exists a need to identify and characterize nucleic acidmolecules encoding 2,5-DKG permeases, so that permeases withadvantageous properties can be used in the commercial production ofascorbic acid and in other important applications. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides an isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity. In one embodiment, theisolated nucleic acid molecule contains a nucleotide sequence having atleast 40% identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:1, 3, 5, 7, 9 and 11. In another embodiment,the isolated nucleic acid molecule contains a nucleotide sequence whichencodes a polypeptide having at least 40% identity to an amino acidsequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10and 12.

Also provided are vectors and cells containing isolated nucleic acidmolecules encoding polypeptides having 2,5-KDG permease activity. In oneembodiment, the cells are bacterial cells selected from the generaPantoea and Klebsiella.

The invention also provides methods of identifying and isolating nucleicacid molecules encoding polypeptides which have 2,5-DKG permeaseactivity. Also provided are methods of enhancing 2-KLG production, byexpressing the nucleic acid molecules of the invention in suitablebacterial cells.

Further provided are isolated polypeptides having 2,5-DKG permeaseactivity, and immunogenic peptides therefrom. The invention alsoprovides antibodies specific for such polypeptides and peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows an alignment of the amino acid sequences of the2,5-DKG permeases designated YiaX2 (SEQ ID NO:12); PE1 (SEQ ID NO:2);PE6 (SEQ ID NO:4); prmA (SEQ ID NO:8); prmB (SEQ ID NO:10) and PK1 (SEQID NO:6).

FIG. 2 shows the biosynthetic pathway from glucose to 2-KLG in abacterial strain suitable for biofermentation.

FIG. 3 shows the metabolic selection strategy used to identify novel2,5-DKG permeases.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel nucleic acid molecules encodingpolypeptides having 2,5-DKG permease activity, and related products andmethods. The molecules of the invention can advantageously be used toincrease the efficiency of 2-KLG bioproduction, and thus to lower thecost of commercial ascorbic acid production.

Naturally occurring 2,5-DKG permeases are polypeptides localized to thecytoplasmic membrane of microorganisms, which are predicted, usingcommercially available topology prediction programs, to contain about 10to 12 transmembrane domains. Each transmembrane spanning segment isabout 20 amino acids in length, with the intracellular and extracellularloops ranging from about 2 to about 83 amino acids in length. Generally,the loop between the fifth and sixth transmembrane domain spanningsegments is larger than the other loops.

Naturally occurring 2,5-DKG permeases are typically about 350–550 aminoacids in length, such as about 400–450 amino acids in length, andparticularly about 425–440 amino acids in length.

The nucleotide sequences encoding six exemplary 2,5-DKG permeases areset forth as follows, with the designation and organismal source of themolecule indicated in parentheses: SEQ ID NO:1 (PE1 from anenvironmental source); SEQ ID NO:3 (PE6 from an environmental source);SEQ ID NO:5 (PK1 from Klebsiella oxytoca); SEQ ID NO:7 (prmA fromPantoea citrea); SEQ ID NO:9 (prmB from Pantoea citrea); and SEQ IDNO:11 (YiaX2 from Klebsiella oxytoca). The corresponding encoded 2,5-DKGpermease amino acid sequences are set forth as SEQ ID NO:2 (PE1); SEQ IDNO:4 (PE6); SEQ ID NO:6 (PK1); SEQ ID NO:8 (prmA); SEQ ID NO:10 (prmB);and SEQ ID NO:12 (YiaX2).

2,5-DKG permeases from different microorganisms exhibit extensive aminoacid sequence relatedness over their entire length, as is evidenced bythe six-way sequence alignment shown in FIG. 1. The overall identity ofthe six permeases shown in FIG. 1 is about 17%, and the overallsimilarity, taking into account conservative substitutions, is about43%.

Based on their predicted topological and sequence similarity, 2,5-DKGpermeases disclosed herein can be further subdivided into two structuralfamilies. The three 2,5-DKG permeases designated YiaX2, PE6 and PrmA arerepresentative of one family of permeases, sharing about 50% overallidentity in a three-way amino acid sequence alignment. The three 2,5-DKGpermeases designated PK1, PE1 and PrmB are representative of a secondfamily of related permeases, sharing about 60% overall identity in athree-way amino acid sequence alignment.

Naturally occurring 2,5-DKG permeases also exhibit 2,5-DKG permeaseactivity. The term “2,5-DKG permease activity,” as used herein, refersto the ability of the polypeptide, when expressed in its nativeorientation at the cell membrane, to transport 2,5-DKG across thecytoplasmic membrane, in comparison with an unrelated controlpolypeptide. Such transport can be either unidirectional orbidirectional.

2,5-DKG permease activity can be determined by a variety of methods. Forexample, 2,5-DKG permease activity can be determined using a metabolicselection assay, as described further in the Example, below. Briefly, abacterial cell either naturally deficient in 2,5-DKG permease activity,or made deficient in 2,5-DKG permease activity, is identified orproduced. As described in the Example, bacterial cells can be madedeficient in endogenous 2,5-DKG permease activity by preparing adeletion mutant of one or more endogenous 2,5-DKG permease genes, usingthe polymerase chain reaction, following methods known in the art. Theterm “deficient,” as used in relation to a cell deficient in2,5-DKG-permease activity, is intended to refer to endogenous 2,5-DKGpermease activity that is comparable to, or less than, the endogenouspermease activity of a K. oxytoca strain deleted in the yiaX2 gene, suchas the strain K. oxytoca ΔyiaX2 [tkr idnO], as assessed either by agrowth assay or by a 2,5-DKG uptake assay.

A cell useful in a metabolic selection assay to determine 2,5-DKGpermease activity of an expressed polypeptide can further naturally becapable of converting intracellular 2,5-DKG to carbon and energy, ormade capable of such conversion by recombinant expression of appropriatemetabolic enzymes. As described in the Example, a combination of nucleicacid molecules encoding a 2-keto-reductase (tkr) and a 5-keto-reductase(idnO), from any bacterial species, can be expressed in the cell, whichtogether provide the cell with the ability to catalyze the reduction of2,5-DKG to gluconic acid. Gluconic acid can then be used by the cell asa carbon and energy source that supports cell growth.

An exemplary bacterial cell suitable for metabolic assays to determine2,5-DKG permease activity is the strain K. oxytoca ΔyiaX2 [tkr idnO]shown in FIG. 3 and described in the Example, below. This strain has adeleted yiaX2 2,5-DKG permease gene, and also recombinantly expressesthe tkr/idnD/idnO operon set forth as SEQ ID NO:13 on a high copy numberplasmid. Within SEQ ID NO:13, nucleotides 292–1236 encode a2-keto-reductase (tkr) (SEQ ID NO:14); nucleotides 1252–2280 encode anidonic acid dehydrogenase (idnD) (SEQ ID NO:15); and nucleotides2293–3045 encode a 5-keto-reductase (idnO) (SEQ ID NO:16).Alternatively, nucleic acid molecules encoding polypeptides whichcontain modifications from the amino acid sequences designated SEQ IDNO:14 or 16, but which retain 2-keto-reductase activity or5-keto-reductase activity, respectively, can be used in metabolicassays. Exemplary amino acid sequences have at least 60%, such as atleast 70%, preferably 80%, 90%, 95% or greater identity to SEQ ID NOS:14or 16, respectively.

The ability of such a bacterial cell to grow on medium containing2,5-DKG as the sole carbon source, upon expression of a candidate2,5-DKG permease, is a measure of the ability of the expressed permeaseto transport 2,5-DKG into the cell, and is thus a measure of its 2,5-DKGpermease activity. Each of SEQ ID NOS:2, 4, 6, 8, 10 and 12 wasdemonstrated to have 2,5-DKG permease activity, as evidenced by theability of K. oxytoca ΔyiaX2 [tkr idno] expressing each permease to growon 2,5-DKG as the sole carbon source.

Likewise, 2,5-DKG permease activity can be determined by measuringuptake of labeled or unlabeled 2,5-DKG. For example, 2,5-DKG can bedetectably labeled, such as with a fluorescent or radioactive tag. Theability of a cell or membrane vesicle expressing a 2,5-DKG permease totake up the detectable label when provided with detectably labeled2,5-DKG, can be determined using detection assays specific for theparticular label, which are well known in the art. Likewise, uptake ofunlabeled 2,5-DKG can be measured by HPLC or other sensitive detectionassay known in the art. Uptake of 2,5-DKG is thus a measure of permeaseactivity. Each of SEQ ID NOS:2, 4, 6, 8, 10 and 12 exhibits 2,5-DKGpermease activity as determined by assay of uptake of radiolabeled2,5-DKG by bacterial cells expressing the recombinant permeases.

Additionally, 2,5-DKG permease activity can be measured in any cell inwhich 2,5-DKG can be converted to a product, by measuring production ofthe product in the presence of extracellular 2,5-DKG. For example, in acell naturally expressing, or recombinantly expressing, a 2,5-DKGreductase, intracellular 2,5-DKG is converted to 2-KLG. The ability ofthe bacterial cell to produce 2-KLG when provided with extracellular2,5-DKG, upon expression of a 2,5-DKG permease, is a measure of theability of the expressed permease to transport 2,5-DKG into the cell,and is thus a measure of its 2,5-DKG permease activity. Intracellular2-KLG can be detected, for example, using HPLC or other sensitivedetection methods known in the art. Other metabolic products of 2,5-DKGcan also be detected, by similar methods.

It will be appreciated that a variety of alternative assays can be usedto determine 2,5-DKG permease activity. For instance, the change in pHacross a cell or vesicle membrane as 2,5-DKG, an acid, is transportedacross the membrane can be detected. Similarly, a decrease over time inextracellular 2,5-DKG can be determined.

Accordingly, using any of the activity assays described herein, thoseskilled in the art can distinguish between a polypeptide having 2,5-DKGpermease activity, and a polypeptide not having such activity.

A 2,5-DKG permease of the invention can selectively transport 2,5-DKG.As used herein in relation to transport activity, the term “selective”refers to preferential transport of 2,5-DKG rather than 2-KLG into orout of the cell. A permease that selectively transports 2,5-DKG willtransport 2,5-DKG at least 2-fold, such as at least 5-fold, includinggreater than 10-fold more efficiently than it transports 2-KLG. Apermease that selectively transports 2,5-DKG is particularlyadvantageous in applications where it is desirable to increaseintracellular production of 2-KLG, such as in the commercial productionof ascorbic acid. In particular, employing a permease that selectivelytransports 2,5-DKG prevents intracellular 2-KLG from competing withextracellular 2,5-DKG for permease-mediated transport through themembrane, and increases the overall efficiency of intracellular 2-KLGproduction.

It will be appreciated that the assays described above for determining2,5-DKG permease activity can be modified to simultaneously, orseparately, determine 2-KLG permease activity. For example, a metabolicassay can be designed in which a bacterial cell can convert eitherintracellular 2,5-DKG or 2-KLG to carbon and energy. In such a cell, therelative ability of the cell to grow on 2,5-DKG as the sole carbonsource, compared with its ability to grow on 2-KLG as the sole carbonsource, is a measure of the ability of the expressed permease toselectively transport 2,5-DKG. Using such an assay, it was determinedthat the 2,5-DKG permeases designated YiaX2, PE1, PE6, prmA and prmB arenon-selective for 2,5-DKG, as they also efficiently catalyze thetransport of 2-KLG, as K. oxytoca ΔyiaX2 [tkr idnO] cells expressingsuch permeases grow well on either 2,5-DKG or 2-KLG. In contrast, PK1selectively transports 2,5-DKG, and K. oxytoca ΔyiaX2 [tkr idnO] cellsexpressing PK1 (SEQ ID NO:6) grow on 2,5-DKG but not on 2-KLG as thesole carbon source.

The invention provides an isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity. The invention nucleicacid molecules of the invention are suitable for a variety of commercialand research applications. For example, one or more of the inventionnucleic acid molecules can be expressed in bacterial cells in order toenhance the rate of uptake of 2,5-DKG by the cells. Enhancing uptake of2,5-DKG has a variety of applications, such as in commercial productionof 2,5-DKG itself, or commercial production of any metabolic product of2,5-DKG. For example, 2,5-DKG uptake is a rate limiting step in thebiosynthesis of 2-KLG, which is a stable intermediate in the synthesisof ascorbic acid. 2-KLG can thus be obtained from bacterial cellsexpressing 2,5-DKG permeases, and converted to ascorbic acid.

Additionally, the invention nucleic acid molecules can be used as probesor primers to identify and isolate 2,5-DKG permease homologs fromadditional species, or as templates for the production of mutantpermeases, using methods known in the art and described further below.Such permeases can have advantageous properties compared with the2,5-DKG permeases disclosed herein as SEQ ID NOS:2, 4, 6, 8, 10 and 12,such as greater enzymatic activity or greater 2,5-DKG selectivity.

In one embodiment, an isolated nucleic acid molecule of the invention isnot completely contained within the nucleotide sequence designated SEQID NO:19 of WO 00/22170, which is the K. oxytoca yia operon. In anotherembodiment, the isolated nucleic acid molecule of the invention is notcompletely contained within the nucleotide sequence herein designatedSEQ ID NO:11. In another embodiment, the encoded polypeptide is notcompletely contained within the amino acid sequence herein designatedSEQ ID NO:12.

The term “isolated,” as used herein, is intended to mean that themolecule is altered, by the hand of man, from how it is found in itsnatural environment. For example, an isolated nucleic acid molecule canbe a molecule operatively linked to an exogenous nucleic acid sequence.An isolated nucleic acid molecule can also be a molecule removed fromsome or all of its normal flanking nucleic acid sequences, such asremoved from one or more other genes within the operon in which thenucleic acid molecule is normally found.

Specifically with respect to an isolated nucleic acid moleculecontaining the nucleotide sequence designated SEQ ID NO:11, or encodingthe yiaX2 polypeptide designated SEQ ID NO:12, the term “isolated” isintended to mean that the nucleic acid molecule does not contain any ofthe flanking open reading frames (orfs) present in the K. oxytoca yiaoperon, such as the orfs designated lyxK and orf1, described in WO00/22170.

An isolated molecule can alternatively, or additionally, be a“substantially pure” molecule, in that the molecule is at least 60%,70%, 80%, 90 or 95% free from cellular components with which it isnaturally associated. An isolated nucleic acid molecule can be in anyform, such as in a buffered solution, a suspension, a heterologous cell,a lyophilized powder, or attached to a solid support.

The term “nucleic acid molecule” as used herein refers to apolynucleotide of natural or synthetic origin. A nucleic acid moleculecan be single- or double-stranded genomic DNA, cDNA or RNA, andrepresent either the sense or antisense strand or both. A nucleic acidmolecule can thus correspond to the recited sequence, to its complement,or both.

The term “nucleic acid molecule” is intended to include nucleic acidmolecules that contain one or more non-natural nucleotides, such asnucleotides having modifications to the base, the sugar, or thephosphate portion, or having one or more non-natural linkages, such asphosphothioate linkages. Such modifications can be advantageous inincreasing the stability of the nucleic acid molecule, particularly whenused in hybridization applications.

Furthermore, the term “nucleic acid molecule” is intended to includenucleic acid molecules modified to contain a detectable moiety, such asa radiolabel, a fluorochrome, a ferromagnetic substance, a luminescenttag or a detectable binding agent such as biotin. Nucleic acid moleculescontaining such moieties are useful as probes for detecting the presenceor expression of a 2,5-DKG permease nucleic acid molecule.

In one embodiment, the isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity contains a nucleotidesequence comprising nucleotides 1–20, 1–100, 101–120, 101–200, 201–220,201–300, 301–320, 301–400, 401–420, 401–500, 501–520, 501–600, 601–620,601–700, 701–720, 701–800, 801–820, 801–900, 901–920, 901–1000,1001–1020, 1001–1100, 1101–1120, 1100–1200, 1201–1220, 1201–1300,1301–1320, 1301–1400, 1401–1420 or 1401–1500 of any of SEQ ID NOS:1, 3,5, 7, 9 or 11.

In another embodiment, the isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity encodes an amino acidsequence comprising amino acids 1–10, 1–50, 51–60, 51–100, 101–110,101–150, 151–160, 151–200, 201–210, 201–250, 251–260, 251–300, 301–310,301–350, 351–361, 351–400, 401–410 or 401–439 of any of SEQ ID NOS:2, 4,6, 8, 10 or 12.

In one embodiment, the isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity contains a nucleotidesequence having at least 35% identity to any of the 2,5-DKG permeasenucleic acid molecules designated SEQ ID NOS:1, 3, 5, 7, 9 or 11.Preferably, such a molecule will have at least 40% identity to any ofthese recited SEQ ID NOS, such as at least 45%, 50%, 60%, 70% or 80%identity, including at least 90%, 95%, 98%, 99% or greater identity toSEQ ID NOS:1, 3, 5, 7, 9 or 11.

In another embodiment, the isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity contains a nucleotidesequence which encodes a polypeptide having at least 35% identity to anyof the 2,5-DKG permease polypeptides designated SEQ ID NOS:2, 4, 6, 8,10 or 12. Preferably, the encoded polypeptide will have at least 40%identity to any of these recited SEQ ID NOS, such as at least 45%, 50%,60%, 70%, 80% identity, including at least 90%, 95%, 98%, 99% or greateridentity to SEQ ID NOS:2, 4, 6, 8, 10 or 12.

The term “percent identity” with respect to a nucleic acid molecule orpolypeptide of the invention is intended to refer to the number ofidentical nucleotide or amino acid residues between the aligned portionsof two sequences, expressed as a percent of the total number of alignedresidues, as determined by comparing the entire sequences using aCLUSTAL V computer alignment and default parameters. CLUSTAL Valignments are described in Higgens, Methods Mol. Biol. 25:307–318(1994), and an exemplary CLUSTAL V alignment of 2,5-DKG permease aminoacid sequences is presented in FIG. 1.

Due to the degeneracy of the genetic code, the nucleotide sequence of anative nucleic acid molecule can be modified and still encode anidentical or substantially similar polypeptide. Thus, degeneratevariants of SEQ ID NOS:1, 3, 5, 7, 9 or 11 are exemplary inventionnucleic acid molecules encoding polypeptides having 2,5-DKG permeaseactivity.

Additionally, nucleic acid molecules encoding 2,5-DKG permeases fromother species of microorganisms are exemplary invention nucleic acidmolecules. The six permeases designated YiaX2, PE1, PE6, prmA, prmB andPK1, which were isolated from at least three, and likely four, differentspecies of microorganisms, share substantial nucleotide sequenceidentity. For example, the two most similar of the disclosed 2,5-DKGpermease nucleotide sequences, SEQ ID NO:5 (PK1) and SEQ ID NO:1 (PE1),share 86% identity across their length. The two most dissimilar of thedisclosed 2,5-DKG permease nucleotide sequences, SEQ ID NO:7 (prmA) andSEQ ID NO:9 (prmB), share 51% identity across their length. In contrast,a search of GenBank reveals no other nucleotide sequences, includingsequences which encode transporter proteins and other transmembraneproteins, that exhibit significant identity or similarity to any of thedisclosed 2,5-DKG permease nucleotide sequences over the entire lengthof their sequences.

The six permeases disclosed herein also share substantial amino acidsequence identity over their entire length, as described previously. Forexample, PK1 from Klebsiella oxytoca (SEQ ID NO:6), and PE1, from anenvironmental source (SEQ ID NO:2), are 93% identical at the amino acidlevel. The amino acid sequence in the GenBank database most closelyrelated to a 2,5-DKG permease, which is a putative tartrate transporterfrom Agrobacterium vitis (GenBank Accession U32375 or U25634) is 33%identical to SEQ ID NO:12 (YiaX2), and shares less identity with theother disclosed 2,5-DKG permeases. Other sequences with some degree ofidentity in the GenBank database to the disclosed 2,5-DKG permeaseinclude membrane transporter proteins from a variety of species,including phthalate transporter proteins from B. cepacia (AF152094) andP. putida (D13229); hydroxyphenylacetate transporters from S. dublin(AF144422) and E. coli (Z37980); and probable transporter proteins fromS. coelicolor (AL136503 and AL132991) each of which has about 27% orless identity at the amino acid level to the recited SEQ ID NOS.

In view of the high degree of identity between different 2,5-DKGpermease nucleic acid molecules and encoded polypeptides within a singlespecies and between different microbial species, additional 2,5-DKGpermeases from other species can be readily identified and tested. Thus,nucleic acid molecules of the invention include nucleic acid moleculesthat encode polypeptides having 2,5-DKG permease activity from anymicrobial species. Microorganisms that contain 2,5-DKG permeases can berecognized by their ability to actively transport 2,5-DKG, such thatthey can grow on 2,5-DKG as the sole carbon source, or incorporate2,5-DKG in an uptake assay. Such microorganisms can include, forexample, bacteria, including Archaebacteria, gram positive and gramnegative bacteria; yeast; and fungi.

Exemplary bacteria which contain 2,5-DKG permeases includeProteobacteria, and more specifically Enterobacteria and Pseudomonads(e.g. P. aeruginosa), as described in the Example. ExemplaryEnterobacteria include species from the genera Klebsiella (e.g. K.oxytoca, from which SEQ ID NOS:5 and 11 were obtained) and Pantoea (e.g.P. citrea, from which SEQ ID NOS:7 and 9 were obtained, and P.agglomerans). Sources of such microorganisms include publicrepositories, such as the American Type Culture Collection (ATCC), andcommercial sources. It will be appreciated that the taxonomy andnomenclature of bacterial genera are such that the same or similarstrains are sometimes reported in the literature as having differentnames. For example, Klebsiella oxytoca (e.g. ATCC 13182) hasalternatively been described as Aerobacter aerogenes, Klebsiellaaerogenes and Klebsiella pneumoniae. Likewise, Pantoea agglomerans (e.g.ATCC 21998) has alternatively been described as Erwinia herbicola andAcetomonas albosesamae. The terms “Klebsiella” and “Pantoea,” as usedherein, are intended to refer to the genera of the strains deposited asATCC 13182 and 21998, respectively.

Additionally, microorganisms from which 2,5-DKG permease nucleic acidmolecules can be obtained are microorganisms present in environmentalsamples. For example, the 2,5-DKG permease nucleic acid moleculesdesignated SEQ ID NOS:1 and 3 were obtained from environmental samples.As used herein, the term “environmental sample” refers to a sampleobtained from natural or man-made environments, which generally containsa mixture of microorganisms.

Exemplary environmental samples are samples of soil, sand, freshwater orfreshwater sediments, marine water or marine water sediments, industrialeffluents, hot springs, thermal vents, and the like. Within anenvironmental sample there are likely to be microorganisms that areunidentified, and also microorganisms that are uncultivable. Isolationof invention 2,5-DKG permease molecules of the invention frommicroorganisms present in environmental samples does not require eitheridentification or culturing of the microorganism.

Furthermore, nucleic acid molecules of the invention include nucleicacid molecules encoding amino acid sequences that are modified by one ormore amino acid additions, deletions or substitutions with respect tothe native sequence of SEQ ID NOS:2, 4, 6, 8, 10 or 12. Suchmodifications can be advantageous, for example, in enhancing thestability, expression level, enzymatic activity, or 2,5-DKG selectivityof the permease. If desired, such. modifications can be randomlygenerated, such as by chemical mutagenesis, or directed, such as bysite-directed mutagenesis of a native permease sequence, using methodswell known in the art.

An amino acid sequence that is modified from a native permease aminoacid sequence can include one or more conservative amino acidsubstitutions, such as substitution of an apolar amino acid with anotherapolar amino acid (such as replacement of leucine with an isoleucine,valine, alanine, proline, tryptophan, phenylalanine or methionine);substitution of a charged amino acid with a similarly charged amino acid(such as replacement of a glutamic acid with an aspartic acid, orreplacement of an arginine with a lysine or histidine); or substitutionof an uncharged polar amino acid with another uncharged polar amino acid(such as replacement of a serine with a glycine, threonine, tyrosine,cysteine, asparagine or glutamine). A modified amino acid sequence canalso include one or more nonconservative substitutions without adverselyaffecting the desired biological activity.

Computer programs known in the art can provide guidance in determiningwhich amino acid residues can be substituted without abolishing theenzymatic activity of a 2,5-DKG permease (see, for example, Eroshkin etal., Comput. Appl. Biosci. 9:491–497 (1993)).

Additionally, guidance in modifying amino acid sequences while retainingor enhancing functional activity is provided by aligning homologous2,5-DKG permease polypeptides from various species (see FIG. 1). It iswell known in the art that evolutionarily conserved amino acid residuesand domains are more likely to be important for maintaining biologicalactivity than less well-conserved residues and domains. Thus, it wouldbe expected that substituting a residue which is highly conserved amongthe six 2,5-DKG permeases shown in FIG. 1 (or among the members of thetwo structural families of permeases, defined as SEQ ID NOS:2, 6 and 10,and SEQ ID NOS:4, 8 and 12) with a non-conserved residue may bedeleterious, whereas making the same substitution at a residue whichvaries widely among the different permeases would likely not have asignificant effect on biological activity.

A comparison of the amino acid sequences of PE1 (SEQ ID NO:2), whichtransports both 2,5-DKG and 2-KLG, and PK1 (SEQ ID NO:6), whichselectively transports 2,5-DKG, indicates that the regions responsiblefor 2,5-DKG selectivity must reside in the 7% of amino acids whichdiffer between these two sequences. Therefore, modifying all or some ofthese differing residues in a 2,5-DKG permease to those found in the PK1sequence would be expected to increase 2,5-DKG selectivity of thepermease.

Alignment of the six 2,5-DKG permeases described herein also providesguidance as to regions where additions and deletions are likely to betolerated. For example the N and C termini, and the region around aminoacids 225–250 (based on the numbering of SEQ ID NO:12 (yiaX2)) appear tobe regions that are relatively tolerant of amino acid insertions anddeletions, as evidenced by gaps in the. sequence alignment. Modified2,5-DKG permeases can thus include “tag” sequences at such sites, suchas epitope tags, histidine tags, glutathione-S-transferase (GST) and thelike, or sorting sequences. Such additional sequences can be used, forexample, to facilitate purification or characterization of a recombinant2,5-DKG permease.

It will be appreciated that confirmation that any particular nucleicacid molecule is a nucleic acid molecule of the invention can beobtained by determining the 2,5-DKG permease activity of the encodedpolypeptide, using one or more of the functional assays describedherein.

The invention further provides an isolated nucleic acid moleculeencoding a polypeptide which has 2,5-DKG permease activity, wherein thenucleic acid molecule is operatively linked to a promoter of geneexpression. The term “operatively linked,” as used herein, is intendedto mean that the nucleic acid molecule is positioned with respect toeither the endogenous promoter, or a heterologous promoter, in such amanner that the promoter will direct the transcription of RNA using thenucleic acid molecule as a template.

Methods for operatively linking a nucleic acid to a desired promoter arewell known in the art and include, for example, cloning the nucleic acidinto a vector containing the desired promoter, or appending the promoterto a nucleic acid sequence using PCR. A nucleic acid moleculeoperatively linked to a promoter of RNA transcription can be used toexpress 2,5-DKG transcripts and polypeptides in a desired host cell orin vitro transcription-translation system.

The choice of promoter to operatively link to an invention nucleic acidmolecule will depend on the intended application, and can be determinedby those skilled in the art. For example, if a particular gene productmay be detrimental to a particular host cell, it may be desirable tolink the invention nucleic acid molecule to a regulated promoter, suchthat gene expression can be turned on or off. An exemplary induciblepromoter known in the art is the lacPO promoter/operator, which isrepressed by the lacI^(q) gene product provided by certain host cells,and induced in the presence of 0.01 to 1 mM IPTG (see Example, below).For other applications, weak or strong constitutive promoters may bepreferred.

The invention further provides a vector containing an isolated nucleicacid molecule encoding a polypeptide which has 2,5-DKG permeaseactivity. The vectors of the invention will generally contain elementssuch as a bacterial origin of replication, one or more selectablemarkers, and one or more multiple cloning sites. The choice ofparticular elements to include in a vector will depend on factors suchas the intended host cell or cells; whether expression of the insertedsequence is desired; the desired copy number of the vector; the desiredselection system, and the like. The factors involved in ensuringcompatibility between a host and a vector for different applications arewell known in the art.

In applications in which the vectors will be used for recombinantexpression of the encoded polypeptide, the isolated nucleic acidmolecules will generally be operatively linked to a promoter of geneexpression, as described above, which may be present in the vector or inthe inserted nucleic acid molecule. In cloning and subcloningapplications, however, promoter elements need not be present.

An exemplary vector suitable for both cloning applications and forexpressing 2,5-DKG permeases in different bacterial species is the lowcopy number plasmid pCL1920 described by Lerner et al., Nucleic AcidsRes. 18:4621 (1994), which contains a spectinomycin resistance gene (seeExample, below).

Also provided are cells containing an isolated nucleic acid moleculeencoding a polypeptide which has 2,5-DKG permease activity. The isolatednucleic acid molecule will generally be contained within a vectorcompatible with replication in the particular host cell. However, forcertain applications, incorporation of the nucleic acid molecule intothe bacterial genome will be preferable.

The cells of the invention can be any cells in which a 2,5-DKG permeasewill be expressed and folded into an active conformation. Guidance inchoosing appropriate host cells is provided by identifying cell typeswhich express other functional 10 to 12 transmembrane transporterproteins. For example, 10 to 12 transmembrane transporter proteins arefound in a variety of bacterial species, as well as in yeast (e.g. S.pombe), Arabidopsis, and Drosophila. Therefore, depending on theparticular application for the host cell, a host cell of the inventioncan be a bacterial cell, yeast, Arabidopsis or Drosophila cell.

In a preferred embodiment, the cell is a bacterial cell. The choice ofbacterial cell will depend on the intended application. For example, forroutine subcloning applications, the cell can be any convenientlaboratory strain of bacteria, such as E. coli, which can be transformedwith the isolated nucleic acid molecules and vectors of the invention bymethods well known in the art.

For assessment of encoded 2,5-DKG permease activity, the cell can be abacterial strain suitable for metabolic assays, such as a strain whichendogenously expresses, or which is engineered to express, enzymes thatcatalyze the conversion of 2,5-DKG to essential products. An exemplarystrain suitable for metabolic assays is the K. oxytoca ΔyiaX2 [tkr idnO]strain designated MGK002 [pDF33] described further in the Example,below, which provides for the conversion of intracellular 2,5-DKG togluconic acid, which can be used as a carbon and energy source.

For use in the commercial bioproduction of 2,5-DKG metabolites, the cellcan be a bacterial strain which endogenously expresses, or which isengineered to express, a 2,5-DKG reductase. As described in U.S. Pat.No. 5,032,514, 2,5-DKG reductases are found in genera includingBrevibacterium, Arthrobacter, Micrococcus, Staphylococcus, Pseudomonas,Bacillus, Citrobacter and Corynebacterium. Therefore, a cell of theinvention can be a bacterial cell of any of these genera, or a bacterialcell engineered to express a 2,5-DKG reductase of any of these genera.

A cell able to produce 2,5-DKG metabolites will preferably also be ableto catalyze the extracellular production of 2,5-DKG from an inexpensivecarbon source, such as glucose. An exemplary pathway from D-glucose to2,5-DKG involves the enzymatic conversion of D-glucose to D-gluconicacid (catalyzed by D-glucose dehydrogenase), from D-gluconic acid to2-Keto-D-gluconic acid (catalyzed by D-gluconate dehydrogenase), andfrom 2-Keto-D-gluconic acid to 2,5-DKG (catalyzed by 2-Keto-D-gluconicacid dehydrogenase), as is shown in FIG. 2. These steps can be carriedout by organisms of several genera, including Gluconobacter, Acetobacterand Erwinia (also called Pantoea).

A bacterial cell useful for the production of 2-KLG from D-glucose isthe Pantoea aggolmerans (also referred to as Erwinia herbicola orAcetomonas albosesamae) strain described in U.S. Pat. No. 5,032,514,designated ATCC 21998 ptrp 1–35 tkrAΔ3, or a derivative of this strainwith improved properties. Contemplated improvements to this strain,which can be produced by genetic engineering, include deletion ofenzymes that divert glucose to metabolites other than 2-KLG, such thatyield of 2-KLG is increased. Other contemplated improvements to thisstrain include mutations that provide for improved recovery andpurification of 2-KLG.

The Pantoea strain described in U.S. Pat. No. 5,032,514 recombinantlyexpresses a 2,5-DKG reductase from Corynebacterium (described in U.S.Pat. No. 4,757,012). The strain further contains a mutation that resultsin a non-functional tkrA gene and is thus deficient in 2-keto reductaseactivity. Mutation of the tkrA gene is advantageous in reducingmetabolic diversion of 2-KLG to L-idonic acid, and metabolic diversionof 2,5-DKG to 5-keto-D-gluconate from 2-KLG.

Expression of one or more 2,5-DKG permeases of the invention in suchcells significantly increases overall production of 2-KLG fromD-glucose, which lowers the cost of commercial production of ascorbicacid.

The cells of the invention can contain one, two or more isolated nucleicacid molecules of the invention that encode polypeptides having 2,5-DKGpermease activity. For example, the cell can contain an isolated nucleicacid molecule encoding at least one polypeptide having at least 80%identity to any of SEQ ID NOS:2, 4, 6, 8, 10 or 12, and optionally willcontain two or more such nucleic acid molecules, in any combination.Preferably, at least one such encoded polypeptide selectively transports2,5-DKG.

In a preferred embodiment, a bacterial cell of the invention suitablefor bioproduction of 2-KLG contains an isolated nucleic acid moleculeencoding a polypeptide having at least 95% identity to the 2,5-DKGselective permease designated SEQ ID NO:8 (prmA); and optionally furthercontaining at least one isolated nucleic acid molecule encoding apolypeptide having at least 95%. identity to a 2,5-DKG permease selectedfrom the group consisting of SEQ ID NO:4 (PE6), SEQ ID NO:10 (prmB) andSEQ ID NO:6 (PK1).

The invention also provides a method of enhancing production of 2-KLG.The method consists of culturing a bacterial cell, wherein the cellcontains an isolated nucleic acid molecule encoding a polypeptide whichhas 2,5-DKG permease activity, under conditions wherein the encoded2,5-DKG permease is expressed and intracellular 2,5-DKG is converted to2-KLG. Cells suitable for this purpose, such as the Pantoea straindescribed in U.S. Pat. No. 5,032,514, have been described above.Optionally, the 2-KLG so produced can be chemically or enzymaticallyconverted to a desired product such as ascorbic acid, following methodsknown in the art.

The invention further provides isolated oligonucleotide molecules thatcontain at least 17 contiguous nucleotides from any of the nucleotidesequences referenced as SEQ ID NOS:1, 3, 5, 7, 9 or 11. As used herein,the term “oligonucleotide” refers to a nucleic acid molecule thatcontains at least 17 contiguous nucleotides from the reference sequenceand which may, but need not, encode a functional protein. Thus, anoligonucleotide of the invention can contain at least 18, 19, 20, 22 or25 contiguous nucleotides, such as at least 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 250, 300, 400, 500, 750, 1000 or morecontiguous nucleotides from the reference nucleotide sequence, up to thefull length of the reference nucleotide sequence. The oligonucleotidesof the invention are thus of sufficient length to be useful assequencing primers, PCR primers, hybridization probes or antisensereagents, and can also encode polypeptides having 2,5-DKG permeaseactivity, or immunogenic peptides therefrom. Those skilled in the artcan determine the appropriate length and sequence of an oligonucleotideof the invention for a particular application.

For certain applications, such as for detecting 2,5-DKG expression in acell or library, it will be desirable to use isolated oligonucleotidemolecules of the invention that specifically hybridize to a nucleic acidmolecule encoding a 2,5-DKG permease. The term “specifically hybridize”refers to the ability of a nucleic acid molecule to hybridize, understringent hybridization conditions as described below, to a nucleic acidmolecule that encodes a 2,5-DKG permease, without hybridizing to asubstantial extent under the same conditions with nucleic acid moleculesthat do not encode 2,5-DKG permeases, such as unrelated molecules thatfortuitously contain short regions of identity with a permease sequence.Thus, a nucleic acid molecule that “specifically hybridizes” is of asufficient length and contains sufficient distinguishing sequence from a2,5-DKG permease to be characteristic of the 2,5-DKG permease. Such amolecule will generally hybridize, under stringent conditions, as asingle band on a Northern blot or Southern blot prepared from mRNA of asingle species.

As used herein, the term “stringent conditions” refers to conditionsequivalent to hybridization of a filter-bound nucleic acid molecule to anucleic acid in a solution containing 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing the filter in0.1×SSPE, and 0.1% SDS at 65° C. twice for 30 minutes. Equivalentconditions to the stringent conditions set forth above are well known inthe art, and are described, for example in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1992).

Nucleotide sequences that are characteristic of each of SEQ ID NOS:1, 3,5, 7 or 9, or which are common to two, three or more of SEQ ID NOS:1, 3,5, 7, 9 or 11 can readily be determined by aligning the sequences usinga CLUSTAL V alignment program. Oligonucleotides containing regions whichare common to two or more different 2,5-DKG permease nucleic acidmolecules can advantageously be used as PCR primers or hybridizationprobes to isolate or detect nucleic acid molecules encoding 2,5-DKGpermeases from other species.

The oligonucleotides of the invention can, but need not, encodepolypeptides having 2,5-DKG activity. Thus, the inventionoligonucleotides can contain sequences from the 5′ or 3′ untranslatedregion, or both, of the nucleotide sequences designated SEQ ID NOS:1, 3,5, 7, 9 or 11, or contain coding sequences, or both. As described abovewith respect to the term “nucleic acid molecule,” the inventionoligonucleotides can be derived from either the sense or antisensestrand of the recited SEQ ID NO.

The oligonucleotides of the invention can also advantageously be used todirect the incorporation of amino acid additions, deletions orsubstitutions into a recombinant 2,5-DKG permease. In such applications,it will be understood that the invention oligonucleotides can containnucleotide modifications with respect to SEQ ID NOS:1, 3, 5, 7, 9 or 11such that the oligonucleotides encode the desired amino acidmodifications to SEQ ID NOS:2, 4, 6, 8, 10 or 12, so long as theycontain at least 17 contiguous residues from the reference sequence.

Exemplary oligonucleotides of the invention are oligonucleotides thatcontain a sequence selected from nucleotides 1–20, 1–100, 101–120,101–200, 201–220, 201–300, 301–320, 301–400, 401–420, 401–500, 501–520,501–600, 601–620, 601–700, 701–720, 701–800, 801–820, 801–900, 901–920,901–1000, 1001–1020, 1001–1100, 1101–1120, 1100–1200, 1201–1220,1201–1300, 1301–1320, 1301–1400, 1401–1420 or 1401–1500 of any of SEQ IDNOS:1, 3, 5, 7, 9 or 11.

The invention further provides a kit containing a pair of 2,5-DKGpermease oligonucleotides packaged together, either in a singlecontainer or separate containers. The pair of oligonucleotides arepreferably suitable for use in PCR applications for detecting oramplifying a nucleic acid molecule encoding a 2,5-DKG permease. The kitcan further contain written instructions for use of the primer pair inPCR applications, or solutions and buffers suitable for suchapplications.

The invention further provides isolated oligonucleotides that contain anucleotide sequence encoding a peptide having at least 10 contiguousamino acids of an amino acid selected from the group consisting of SEQID NOS:2, 4, 6, 8, 10 or 12. Such oligonucleotides can encode at least10, 12, 15, 20, 25 or more contiguous amino acids of SEQ ID NOS:2, 4, 6,8, 10 or 12, such as at least 30, 40, 50, 75, 100, 200, 300, 400 or morecontiguous amino acids from the reference sequence. The encoded peptidescan be expressed from such oligonucleotides, by routine methods, andused to produce, purify or characterize 2,5-DKG antibodies, as will bediscussed further below. The peptides encoded by such oligonucleotidescan, but need not, additionally have 2,5-DKG permease enzymaticactivity.

In one embodiment, the isolated oligonucleotide encodes an amino acidsequence selected from amino acids 1–10, 1–50, 51–60, 51–100, 101–110,101–150, 151–160, 151–200, 201–210, 201–250, 251–260, 251–300, 301–310,301–350, 351–361, 351–400, 401–410, 401–439 of any of SEQ ID NOS:2, 4,6, 8, 10 or 12.

Isolated nucleic acid molecules which encode polypeptides having 2,5-DKGpermease activity, as well as the isolated oligonucleotides describedabove, will be subsequently referred as “2,5-DKG permease nucleic acidmolecules.”

The isolated 2,5-DKG permease nucleic acid molecules of the inventioncan be prepared by methods known in the art. The method chosen willdepend on factors such as the type and size of nucleic acid molecule oneintends to isolate; whether or not it encodes a biologically activepolypeptide (e.g. a polypeptide having permease activity orimmunogenicity); and the source of the nucleic acid molecule. Thoseskilled in the art can isolate or prepare 2,5-DKG permease nucleic acidmolecules as genomic DNA or desired fragments therefrom; as full-lengthcDNA or desired fragments therefrom; or as full-length MRNA or desiredfragments therefrom, from any microorganism of interest.

An exemplary method of preparing a 2,5-DKG permease nucleic acidmolecule is by isolating a recombinant construct which encodes andexpresses a polypeptide having 2,5-DKG permease activity. As describedin the Example, one useful method is to provide a metabolic selectionsystem where bacterial cell growth is made dependent on expression of a2,5-DKG permease, introducing expressible DNA, such as a cDNA or genomiclibrary, into the assay cells, selecting surviving cells under theselective conditions, and isolating the introduced DNA. Alternatively, ascreening method can be designed, such that a cell will exhibit adetectable signal only when expressing a functional 2,5-DKG permease. Anexemplary detectable signal is intracellular incorporation of adetectable label present on 2,5-DKG. Additionaly screening and selectionstrategies suitable for identifying nucleic acid molecules encodingmetabolic enzymes are described, for example, in PCT publication WO00/22170 and U.S. Pat. Nos. 5,958,672 and 5,783,431.

A further method for producing an isolated 2,5-DKG permease nucleic acidmolecule involves amplification of the nucleic acid molecule using2,5-DKG permease-specific primers and the polymerase chain reaction(PCR). Using PCR, a 2,5-DKG permease nucleic acid molecule having anydesired boundaries can be amplified exponentially starting from aslittle as a single gene or mRNA copy, from any cell having a 2,5-DKGpermease gene.

Given the high degree of identity among the six disclosed 2,5-DKGpermeases, those skilled in the art can design suitable primers forisolating additional 2,5-DKG permease nucleic acid molecules. Suchprimers are preferably degenerate oligonucleotides that encode, or arecomplementary to, short consensus amino acid sequences present in two ormore of the 2,5-DKG permeases disclosed herein, such as oligonucleotidesthat encode 10 or more contiguous amino acids present in at least two ofSEQ ID NOS: 2, 6 and 10, or oligonucleotides that encode 10 or morecontiguous amino acids present in at least two of SEQ ID NOS:4, 8 and12. Such sequences can be determined from an alignment of amino acidsequences shown in FIG. 1. Exemplary amino acid sequences present in atleast two of SEQ ID NOS:2, 6 and 10 are amino acids 19–31, 115–124,146–156, and 339–348 of SEQ ID NO:2. Exemplary amino acid sequencespresent in at least two of SEQ ID NOS:4, 8 and 12 are amino acids 55–64,60–69, 252–261, and-370–379 of SEQ ID NO:8.

Methods are well known in the art to determine or modify PCR reactionconditions when using degenerate primers to isolate a desired nucleicacid molecule. The amplified product can subsequently be sequenced, usedas a hybridization probe, or used for 5′ or 3′ RACE to isolate flankingsequences, following procedures well known in the art and described, forexample, in Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York (2000).

Given the high degree of sequence identity and structural relatednessamong the six disclosed 2,5-DKG permeases, homologs from any otherspecies can readily be identified by either hybridization or antibodyscreening. For example, an isolated 2,5-DKG permease nucleic acidmolecule can be identified by screening a library, such as a genomiclibrary, cDNA library or expression library, with a detectable nucleicacid molecule or antibody. Such libraries are commercially availablefrom a variety of microorganisms, or can be produced from any availablemicroorganism or environmental sample of interest using methodsdescribed, for example, in PCT publication WO 00/22170. The libraryclones identified as containing 2,5-DKG permease nucleic acid moleculescan be isolated, subcloned and sequenced by routine methods.

Furthermore, 2,5-DKG permease nucleic acid molecules can be produced bydirect synthetic methods. For example, a single stranded nucleic acidmolecule can be chemically synthesized in one piece, or in severalpieces, by automated synthesis methods known in the art. Thecomplementary strand can likewise be synthesized in one or more pieces,and a double-stranded molecule made by annealing the complementarystrands. Direct synthesis is particularly advantageous for producingrelatively short molecules, such as oligonucleotide probes and primers,and also for producing nucleic acid molecules containing modifiednucleotides or linkages.

The invention also provides an isolated polypeptide which has 2,5-DKGpermease activity. Such isolated polypeptides, when expressed in theirnormal configuration at the cell membrane, are useful in applications inwhich enhanced uptake of 2,5-DKG is desirable, such as in bioproductionof 2-KLG. The isolated polypeptides of the invention can also be addedto a culture medium, preferably in a membrane vesicle, to compete withmembrane-bound permeases for 2,5-DKG, and thus to stop 2,5-DKG uptake.Thus, isolated polypeptides having 2,5-DKG permease activity can be usedto regulate production of 2,5-DKG metabolites.

In one embodiment, an isolated polypeptide of the invention is notencoded by a nucleotide sequence completely contained within thenucleotide sequence designated SEQ ID NO:19 of WO 00/22170, which is theK. oxytoca yia operon. In another embodiment, an isolated polypeptide ofthe invention is not completely contained within the amino acid sequenceherein designated SEQ ID NO:12.

An “isolated” polypeptide of the invention is altered by the hand of manfrom how it is found in its natural environment. For example, anisolated 2,5-DKG permease can be a molecule that is recombinantlyexpressed, such that it is present at a higher level in its native host,or is present in a different host. Alternatively, an “isolated” 2,5-DKGpermease of the invention can be a substantially purified molecule.Substantially purified 2,5-DKG permeases can be prepared by methodsknown in the art. Specifically with respect to a polypeptide encodingthe yiaX2 polypeptide designated SEQ ID NO:12, the term “isolated” isintended to mean that polypeptide is not present in association with thepolypeptides expressed by other genes in the K. oxytoca yia operon, suchas the genes, designated lyxK and orf1, described in WO 00/22170.

In one embodiment, an isolated polypeptide having. 2,5-DKG permeaseactivity contains an amino acid sequence having at least 40% identity toan amino acid sequence selected from the group consisting of SEQ IDNOS:2, 4, 6, 8, 10 or 12. Preferably, the encoded polypeptide will haveat least 45% identity to any of the recited SEQ ID NOS, such as at least50%, 60%, 70%, 80% identity, including at least 90%, 95%, 98%, 99% orgreater identity.

In another embodiment, the isolated polypeptide having 2,5-DKG permeaseactivity contains at least 10 contiguous amino acids of any of SEQ IDNOS:2, 4, 6, 8, 10 or 12. Exemplary invention polypeptides contain anamino acid sequence of amino acids 1–10, 1–50, 51–60, 51–100, 101–110,101–150, 151–160, 151–200, 201–210, 201–250, 251–260, 251–300, 301–310,301–350, 351–361, 351–400, 401–410, 401–439 of any of SEQ ID NOS:2, 4,6, 8, 10 or 12.

Also provided is an isolated immunogenic peptide having an amino acidsequence derived from a 2,5-DKG permease. Such isolated immunogenicpeptides are useful, for example, in preparing and purifying 2,5-DKGantibodies. The term “immunogenic,” as used herein, refers to a peptidethat either is capable of inducing 2,5-DKG permease-specific antibodies,or capable of competing with 2,5-DKG permease-specific antibodies forbinding to a 2,5-DKG permease. Peptides that are likely to beimmunogenic can be predicted using methods and algorithms known in theart and described, for example, by Irnaten et al., Protein Eng.11:949–955 (1998), and Savoie et al., Pac. Symp. Biocomput. 1999:182–189(1999). The immunogenicity of the peptides of the invention can beconfirmed by methods known in the art, such as by delayed-typehypersensitivity response assays in an animal sensitized to a 2,5-DKGpermease, or by direct or competitive ELISA assays.

An isolated immunogenic peptide of the invention can contain at least 10contiguous amino acids of a polypeptide selected from the groupconsisting of SEQ ID NOS:2, 4, 6, 8, 10 or 12, such as amino acids 1–10,1–50, 51–60, 51–100, 101–110, 101–150, 151–160, 151–200, 201–210,201–250, 251–260, 251–300, 301–310, 301–350, 351–361, 351–400, 401–410,401–439 of any of SEQ ID NOS:2, 4, 6, 8, 10 or 12. Such a peptide canhave at least 12, 15, 20, 25 or more contiguous amino acids of thereference sequence, including at least 30, 40, 50, 75, 100, 200, 300,400 or more contiguous amino acids from the reference sequence, up tothe full-length sequence.

For the production of antibodies that recognize 2,5-DKG permeases intheir native configuration, such peptides will preferably contain atleast part of an extracellular or intracellular domain from thepermease. An extracellular or intracellular domain is generallycharacterized by containing at least one polar or positively ornegatively charged residue, whereas a transmembrane domain is generallycharacterized as an uninterrupted stretch of about 20 contiguoushydrophobic residues. Commercially available computer topology programscan be used to determine whether a peptide is likely to correspond to anextracellular or intracellular domain or to a transmembrane region.Immunogenic peptides of the invention derived from a transmembraneregion are useful to raise antibodies for use in applications such asimmunoblotting, where the 2,5-DKG polypeptide need not be in its nativeconfiguration to be recognized.

The structural and functional characteristics and applications of2,5-DKG permease polypeptides of the invention have been described abovewith respect to the encoding nucleic acid molecules, and are equallyapplicable in reference to the isolated polypeptides of the invention.Isolated polypeptides having 2,5-DKG permease activity, as well as theisolated immunogenic peptides of the invention, will subsequently bereferred to as “2,5-DKG permeases.”

Methods for recombinantly producing 2,5-DKG permeases have beendescribed above with respect to nucleic acid molecules, vectors andcells of the invention. 2,5-DKG permeases can alternatively be preparedby biochemical procedures, by isolating membranes from bacteria thatnaturally express, or recombinantly express, 2,5-DKG permeases. Themembranes can be further fractionated by size or affinitychromatography, electrophoresis, or immunoaffinity procedures, toachieve the desired degree of purity. Purification can be monitored by avariety of procedures, such as by immunoreactivity with 2,5-DKG permeaseantibodies, or by a functional assay.

Immunogenic peptides can be produced from purified or partially purified2,5-DKG permease polypeptides, for example, by enzymatic or chemicalcleavage of the full-length polypeptide. Methods for enzymatic andchemical cleavage and for purification of the resultant peptidefragments are well known in the art (see, for example, Deutscher,Methods in Enzymology, Vol. 182, “Guide to Protein Purification,” SanDiego: Academic Press, Inc. (1990)).

Alternatively, 2,5-DKG permeases can be produced by chemical synthesis.If desired, such as to optimize their functional activity, stability orbioavailability, such chemically synthesized molecules can includeD-stereoisomers, non-naturally occurring amino acids, and amino acidanalogs and mimetics. Sawyer, Peptide Based Drug Design, ACS, Washington(1995) and Gross and Meienhofer, The Peptides: Analysis. Synthesis,Biology, Academic Press, Inc., New York (1983). For certainapplications, such as for detecting the polypeptide, it can also beuseful to incorporate one or more detectably labeled amino acids into achemically synthesized permease, such as radiolabeled or fluorescentlylabeled amino acids.

An isolated 2,5-DKG permease of the invention can further be conjugatedto carrier molecules, such as keyhole lympet hemocyanin, which canenhance recognition by the immune system of the isolated 2,5-DKGpermease for production of antibodies. For certain applications, such asto increase the stability or bioactivity of the molecule, or tofacilitate its identification, the 2,5-DKG permease can be chemically orenzymatically derivatized, such as by acylation, phosphorylation orglycosylation.

The invention also provides an antibody specific for a polypeptidehaving 2,5-DKG permease activity, such as an antibody specific for apolypeptide having the amino acid sequence of any of SEQ ID NOS:2, 4, 6,8, 10 or 12. Also provided is an antibody specific for an isolatedpeptide that contains at least 10 contiguous amino acids of any of SEQID NOS:2, 4, 6, 8, 10 or 12, wherein the peptide is immunogenic. Theantibodies of the invention can be used, for example, to detect orisolate 2,5-DKG permeases from expression libraries or cells.

The term “antibody,”as used herein, is intended to include moleculeshaving specific binding activity for a 2,5-DKG permease of at leastabout 1×10⁵ M⁻¹, preferably at least 1×10⁷ M⁻¹, more preferably at least1×10⁹ M⁻¹. The term “antibody” includes both polyclonal and monoclonalantibodies, as well as antigen binding fragments of such antibodies(e.g. Fab, F(ab′)₂, Fd and Fv fragments and the like). In addition, theterm “antibody” is intended to encompass non-naturally occurringantibodies, including, for example, single chain antibodies, chimericantibodies, bifunctional antibodies, CDR-grafted antibodies andhumanized antibodies, as well as antigen-binding fragments thereof.

Methods of preparing and isolating antibodies, including polyclonal andmonoclonal antibodies, using peptide and polypeptide immunogens, arewell known to those skilled in the art and are described, for example,in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1988). Non-naturally occurring antibodies can beconstructed using solid phase peptide synthesis, can be producedrecombinantly or can be obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains. Such methods are described, for example, in Huse et al.Science 246:1275–1281(1989); Winter and Harris, Immunol. Today14:243–246 (1993); Ward et al., Nature 341:544–546 (1989); Hilyard etal., Protein Engineering: A practical approach (IRL Press 1992); andBorrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995).

The following example is intended to illustrate but not limit thepresent invention.

EXAMPLE

This example shows the isolation and characterization of nucleic acidmolecules encoding six novel polypeptides having 2,5-DKG permeaseactivity.

Identification of YiaX2 as a 2,5-DKG Permease

WO 002170 describes the identification and sequencing of an operon fromKlebsiella oxytoca, designated the yia operon, which contains eightputative open reading frames. Because disruption of this operonabolished the ability of K. oxytoca to utilize ascorbic acid as the solecarbon source, the yia operon was predicted to be involved in thecatabolism of ascorbic acid. The functions of the polypeptides encodedby the individual open reading frames in the yia operon were notdescribed in WO 002170.

It was determined that K. oxytoca was able to grow on 2,5-DKG as a solecarbon source and, therefore, it was concluded that K. oxytoca expresseda 2,5-DKG permease. It was predicted that such a permease would sharestructural properties with known bacterial transporter proteins, such asmultiple transmembrane segments. One of the uncharacterized open readingframes in the yia operon, designated yiaX2, encoded a transmembranepolypeptide with about 33% identity to a known tartrate transporter, andwas thus considered a candidate 2,5-DKG permease.

In order to determine whether yiaX2 encoded a 2,5-DKG permease, thisgene was deleted from the chromosome of the K. oxytoca strain designatedM5a1. M5a1 has also been described in the literature as K. pneumonia(see, for example, Streicher et al., Proc. Natl. Acad. Sci. 68:1174–1177(1971)). The yiaX2 deletion mutant was constructed by joining sequencesimmediately upstream and downstream of the yiaX2 gene in a three-wayligation with the pMAK705 integration vector (described in Hamilton etal., J. Bacteriol. 171:4617–4622 (1989)). A fragment of about 1 kb inthe orf1 gene was amplified using oligonucleotides5′-ACCCAAGCTTCACCAAAAGAGTGAAGAGGAAG-3′ (SEQ ID NO:17) and5′-CGTATCTAGAAAAATATTCTGGTGATGAAGGTGA-3 (SEQ ID NO:18), and digestedwith HindIII and XbaI. A fragment of a similar size in the lyxK gene wasamplified with oligonucleotides 5′-AGACTCTAGATCCACATAAACGCACTGCGTAAAC-3′(SEQ ID NO:19) and 5′-GAGGGGATCCTGGCTTCGTGAACGATATACTGG-3′ (SEQ IDNO:20), and digested with XbaI and BamHI. The two resulting fragmentswere ligated together between the HindIII and BamHI sites of the vectorpMAK705. The resulting plasmid was transformed into K. oxytoca strainM5a1, and candidates in which the deletion construct had integrated bydouble crossover were obtained as described in Hamilton et al., supra(1989). The designation of the resulting K. oxytoca ΔyiaX2 strain isMGK002. The yiaX2-deficient phenotype was verified by by PCR analysis.

As described below, the K. oxytoca ΔyiaX2 [tkr idnO] strain wasdetermined to grow very inefficiently on 2,5-DKG as the sole carbonsource, and not to grow on 2-KLG. Confirmation that yiax2 encoded apolypeptide having 2,5-DKG and 2-KLG permease activities was obtained bydetermining that adding back the gene restored the ability of the K.oxytoca ΔyiaX2 [tkr idnO] to grow well on either 2,5-DKG or 2-KLG (seebelow).

Construction of K. Oxytoca ΔYiaX2 [tkr idnO]

In order to identify additional 2,5-DKG permeases, and preferablypermeases selective for 2,5-DKG, a metabolic selection strategy wasutilized. As described in WO 00/22170, metabolic selection isadvantageous in allowing rapid identification of functional genes fromuncharacterized and even unculturable microorganisms, without any priorsequence information.

A tester strain for the metabolic selection of nucleic acid moleculesencoding 2,5-DKG permeases was prepared by engineering K. oxytoca ΔyiaX2to express enzymes involved in the catabolism of 2,5-DKG to gluconicacid, which can be converted to carbon and energy. Enzymes capable ofcatabolizing 2,5-DKG to gluconic acid are encoded by the tkr and idnOgenes of the tkr idnD idnO operon designated SEQ ID NO:13.

The tkr idnD idnO operon (SEQ ID NO:13) was subcloned into the high copynumber vector pUC19 and the resulting clone, designated pDF33, wastransformed into K. oxytoca ΔyiaX2. The resultingtester strain(designated MGK002[pDF33] or K. oxytoca ΔyiaX2 [tkr idnO]) thusexpresses all polypeptides required for the utilization of 2,5-DKG as asole carbon source, but is deficient in 2,5-DKG permease activity totransport extracellular 2,5-DKG into the cell. Therefore, a nucleic acidmolecule that encodes a 2,5-DKG permease, upon expression in the testerstrain, should confer the ability of the tester strain to grow on2,5-DKG. The metabolic selection strategy is shown schematically in FIG.3.

To validate the proposed metabolic selection strategy, as a positivecontrol the yiaX2 gene was reintroduced into the tester strain toconfirm that it conferred the ability to grow on 2,5-DKG and 2-KLG. TheyiaX2 open reading frame (nucleotides 3777 to 5278 of SEQ ID NO:19 of WO00/22170) was PCR-amplified using olignucleotides5′-AATAGGATCCTTCATCACCAGAATATTTTTA-3′ (SEQ ID NO:21) and5′-CATAGGTACCGGCTTTCAGATAGGTGCC-3′ (SEQ ID NO:22) digested with BamH1and Kpn1 and ligated into pCL1920 (Lerner et al., Nucl. Acids. Res.18:4631 (1990); and see description below) previously digested with thesame restriction enzymes. K. oxytoca ΔyiaX2 [tkr idnO], transformed withthe resulting construct, was able to grow overnight at 30° C. on M9minimal agar medium supplemented with either 2-KLG or 2,5-DKG (0.25%)and 0.1 mM IPTG. Therefore, K. oxytoca ΔyiaX2 [tkr idnO] was confirmedto be an appropriate tester strain to identify additional novel 2,5-DKGpermeases, and to determine their selectivity.

Construction of Bacterial Genomic Libraries

The cloning vector used for constructing the above positive control andfor preparing bacterial genomic libraries is plasmid pCL1920 (Lerner etal., supra, 1990), a low-copy number expression vector which carries aspectinomycin/streptomycin resistance determinant. Expression is drivenby the lacPO promoter/operator region which is repressed by the lacI^(q)gene product when provided by the host, and induced in the presence of0.01 to 1 mM IPTG.

Genomic DNA from the following species and isolates was preparedaccording to the method outlined below: Pantoea citrea (ATCC 39140),Klebsiella oxytoca MGK002 (ΔyiaX2), Pseudomonas aeruginosa, and amixture of 25 environmental isolates, obtained from 18 different soiland water samples, and able to grow on 2,5-DKG as the sole carbonsource. Klebsiella oxytoca MGK002 (ΔyiaX2) was among the bacteria chosenbecause there was a slight amount of background growth observable in thetester strain on 2,5-DKG as the sole carbon source, and some 2,5-DKGpermease activity in an uptake assay. However, the tester strain did notgrow on 2-KLG, and exhibited no detectable 2-KLG uptake, suggesting thepresence of a second 2,5-DKG permease with selectivity for 2,5-DKG in K.oxytoca.

Five milliliters of an overnight culture in LB (30° C.) were centrifugedfor 5 min at 6,000 rpm. Pellets were washed with 1.5 ml Tris 10 mM, EDTA1 mM pH 8.0 (TE), centrifuged again and resuspended in 0.4 ml TE.Lysozyme (5 mg/ml) and RNase (100 pg/ml) were added and cells wereincubated for 10 min at 37° C. Sodium dodecylsulfate (SDS) was added toa final concentration of 1% and the tubes were gently shaken until lysiswas complete. One hundred microliters of a 5N NaCl0₄ stock solution wereadded to the lysate. The mixture was extracted once with one volume ofphenol:chloroform (1:1) and once with one volume of chloroform.Chromosomal DNA was precipitated by adding 2 ml of cold (−20° C.)ethanol and gently coiling the precipitate around a curved Pasteurpipette. DNA was dried for 30 min at room temperature and resuspended in50 to 100 μ1 of Tris 10 mM, EDTA 1 mM, NaCl 50 mM pH 8.0 to obtain a DNAconcentration of 0.5 to 1 μg/μl. Genomic DNA preparations from eachenvironmental isolate were mixed in equal ratios to prepare a singlemixed library.

For each preparation, an aliquot of 10–15 μl of genomic DNA wassubjected to Sau3A controlled digestion in order to obtain fragmentsranging between 3 to 20 kb in size. Half that amount was ligated withthe low-copy number expression vector pCL1920, which had previously beendigested with BamHI and dephosphorylated. The resulting genomiclibraries were transformed into E.coli DH10B electrocompetent cells(GIBCO-BRL) and briefly amplified overnight at 30° C. on LB-agarsupplemented with 100 μg/ml spectinomycin. For each library, 30,000 to120,000 clones were plated out and plasmid DNA was bulk-extracted usingstandard procedures. Insert size was randomly checked and the amplifiedlibraries were stored in the form of plasmid DNA at −20° C. for furtheruse in the tester strain.

Selection, Identification and Sequencing of Permease Genes

An aliquot of each genomic library was introduced by electroporationinto the K. oxytoca ΔyiaX2 [tkr idnO] (MGK002[pDF33]) strain. The amountof DNA used in the transformation was adjusted in order to plate out5×10⁵ to 1×10⁶ clones per library on the selective medium. Eachselection round was plated on LB-agar containing 100 μg/mlspectinomycin, then replica-plated onto M9-agar plates containing 2.5%2,5-DKG and 0.1 mM IPTG and adjusted to pH 4.5. The clones that grew on2,5-DKG were transferred into K. oxytoca ΔyiaX2 (MGK002) devoid ofplasmid pDF33, to verify that the tkr idnDO pathway was indeed requiredfor growth of those clones on 2,5-DKG. A brief genetic characterizationwas performed to eliminate identical clones. Following preliminary2,5-DKG/2-KLG uptake assays, 5 clones were retained for furtheranalysis: 2 originated from the Pantoea citrea library, 1 from K.oxytoca and 2 from the mixed environmental library.

In all cases, DNA sequencing of the vector inserts revealed the presenceof a nucleotide sequence (SEQ ID NOS:1, 3, 5, 7 and 9). encoding apolypeptide (SEQ ID NOS:2, 4, 6, 8 and 10) displaying homology withpublished transporters and with yiaX2 (SEQ ID NO:11, and its encodedpolypeptide SEQ ID NO:12). Also present on these inserts were otherorfs, and in most cases an endogenous promoter.

The insert containing both of the prmA and prmB orfs (SEQ ID NOS:7 and9) was about 9 kb, and also contained an orf homologous to bacterialidnO, two orfs encoding transcriptional repressors, an orf of unknownfunction, and 3 orfs encoding homologs of E. coli polypeptides involvedin nitrate utilization.

The insert containing the PE1 orf (SEQ ID NO:1) was about 3 kb, and alsocontained a putative dehydro-deoxygluconokinase gene closely related tothe B. subtilis kdgK gene and a homolog of the E. coli ydcG gene.

The insert containing the PE6 orf (SEQ ID NO:3) was about 6.7 kb. Thegenomic environment of PE6 appeared similar to the yia operon of E. coliand K. oxytoca, as SEQ ID NO:4 was preceded by a yiaL homolog and a yiaKhomolog was also present on the insert.

The insert containing the PK1 orf (SEQ ID NO:5) was about 5.5 kb. Incontrast to the other inserts, this insert did not appear to contain anendogenous promoter, indicating that the PK1 orf was apparentlytranscribed from the vector's promoter. The PK1 orf was directlyfollowed by a tkr homolog.

Nucleic acid molecules encoding each 2,5-DKG permease were reintroducedinto K. oxytoca ΔyiaX2 [tkr idnO] and the resulting strains assayed forgrowth on 2,5-DKG and 2-KLG, and also assayed for uptake of radiolabeled2,5-DKG and 2-KLG.

The uptake assays were performed by mixing radioactive 2-KLG or 2,5-DKGwith IPTG-induced cells, removing aliquots at regular intervals, andmeasuring both the decrease in radioactivity in the supernatant and theappearance of radioactivity in the cells over time. The results of thegrowth assays and 2,5-DKG uptake assay are shown in Table 1, below.

TABLE 1 Recombinantly 2,5-DKG expressed Cell Growth Cell Growth Uptake2,5 DKG Permease on 2,5-DKG on 2-KLG (g/l/h) YiaX2 + ++ 3.7 PE1 ++ ++4.2 PE6 ++ ++ 5.0 prmA ++ ++ 5.5 prmB +/− ND 0.9 prmA and prmB ++ ND 9.9PK1 ++ — 4.2 Control bkgd − 1.0 (K. oxytoca ΔyiaX2/ tkr/idn0)

Nucleic acid molecules encoding the different permeases were alsosubcloned into a variety of vectors, including the high copy numbervector pSE380 (which contains a tac promoter), the medium copy numbervector pACYC184 (which is promoterless), or the low copy number vectorpCL1920, and introduced into a Pantoea strain suitable for bioproductionof 2-KLG from glucose (see U.S. Pat. No. 5,032,514). The resultingstrains were assessed under biofermentation conditions to determinewhich combinations of nucleic acid molecules, promoters and vectors areoptimal for enhancing 2-KLG production.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention.

1. A bacterial cell comprising a first isolated nucleic acid moleculeencoding a polypeptide having 2,5-diketo-D-gluconic acid (2,5-DKG)permease activity and at least 95% sequence identity to SEQ ID NO:12 andsecond isolated nucleic acid molecule encoding a polypeptide having5-keto reductase activity, said polypetide having at least 95% sequenceidentity to SEQ ID NO: 16, wherein said bacterial cell is deficient inendogenous 2,5-DKG permease activity.
 2. A method of enhancing2-keto-L-gluconic acid (2-KLG) production, comprising a) introducing anisolated nucleic acid molecule encoding a polypeptide having at least96% sequence identity to SEQ ID NO: 12 into a bacterial cell whichexpresses an enzyme that catalyzes the conversion of2,5-diketo-D-gluconic acid (2,5-DKG) to 2-KLG, b) allowing expression ofthe polypeptide encoded by said nucleic acid molecule and c) culturingthe bacterial cell under suitable conditions to produce 2-KLG.
 3. Themethod of claim 2, wherein said bacterial cell further expresses enzymesthat catalyze the conversion of glucose to 2,5-DKG.
 4. The method ofclaim 3, wherein said bacterial cell is deficient In endogenous 2-ketoreductase activity.
 5. The method of claim 2, wherein said bacterialcell is of the genus Pantoea.
 6. The method of claim 2, furthercomprising converting said 2-KLG to ascorbic acid.
 7. The bacterial cellof claim 1, which is an E. coli cell.
 8. The method of claim 2, whereinthe nucleic acid molecule has the sequence of SEQ ID NO: 11 or asequence having at least 95% sequence identity thereto.
 9. A method forincreasing the transport of 2,5 diketo-D-gluconic acid (2,5 DKG) acrossa cell membrane into a bacterial host cell comprising a) introducing anisolated nucleic acid molecule into a bacterial host cell, wherein thenucleic acid molecule encodes a protein comprising at least 95% sequenceidentity to SEQ ID NO: 12 and said protein having 2,5 DKG permeaseactivity, b) allowing expression of the protein and c) culturing thebacterial host cell under suitable conditions for the transport of2,5-DKG into the bacterial host cell.
 10. The method according to claim9, wherein the bacterial host cell is an E. coli, Pantoea or Klebsiellahost cell.
 11. The method according to claim 9, wherein the nucleic acidmolecule has the sequence of SEQ ID NO: 11 or a sequence having at least95% sequence identity thereto.
 12. The method according to claim 2,wherein said polypeptide has the sequence of SEQ ID NO:
 12. 13. Themethod according to claim 2, wherein the bacterial host cell is an Ecoli, Pantoea or Klebsiella host cell.
 14. The method according to claim9, wherein said polypeptide has the sequence of SEQ ID NO:
 12. 15. Themethod according to claim 10, wherein the bacterial host cell is aKlebsiella cell.
 16. The method according to claim 10, wherein thebacterial host cell is an E. coli cell.
 17. The method according toclaim 10, wherein the bacterial host cell is a Pantoea cell.
 18. Themethod according to claim 10, wherein the bacterial host cell isdeficient in endogenous 2,5 DKG permease activity.
 19. The methodaccording to claim 10, wherein the bacterial host cell further comprisesa nucleic acid molecule encoding a polypeptide having 2-keto reductaseactivity and at least 95% sequence identity to SEQ ID NO:
 14. 20. Themethod according to claim 10, wherein the bacterial host cell furthercomprises an isolated nucleic acid molecule having 5-keto reductaseactivity and at least 95% sequence identity to SEQ ID NO:
 16. 21. Themethod according to claim 10, wherein the bacterial host cell expressesan enzyme that catalyzes the conversion of 2,5-DKG to 2-keto-L-gluconicacid (2-KLG).
 22. The method according to claim 10, wherein the nucleicacid molecule encoding the protein having 2,5-DKG permease activity isoperably linked to a lac promoter.
 23. The method of claim 2, whereinthe nucleic acid molecule encodes a polypeptide having at least 98%sequence identity to SEQ ID NO:
 12. 24. The method of claim 23, whereinthe nucleic acid molecule encodes a polypeptide having at least 99%sequence identity to SEQ ID NO:
 12. 25. The bacterial cell of claim 1,wherein said cell is from the genus Pantoea.
 26. The bacterial cell ofclaim 1, wherein the first nucleic acid molecule encodes a polypeptidehaving at least 98% sequence identity to SEQ ID NO:12 and the secondnucleic acid encodes a polypeptide having at least 95% sequence identityto SEQ ID NO:16.
 27. The method of claim 9, wherein the nucleic acidmolecule encodes a polypeptide having at least 98% sequence Identity toSEQ ID NO:
 12. 28. The method of claim 27, wherein the nucleic acidmolecule encodes a polypeptide having at least 99% sequence identity toSEQ ID NO: 12.